JP6345186B2 - Use of BT lipopeptide as a therapeutic for obesity and related diseases - Google Patents

Description

This application claims priority from US Provisional Application No. 61 / 738,166, filed Dec. 17, 2012, which is hereby incorporated by reference in its entirety.

  The present invention relates generally to the field of biotechnology, and more specifically to the use of the lipopeptide family to treat or prevent obesity-related diseases including obesity and type 2 diabetes.

  Obesity causes a serious group of diseases known collectively as metabolic syndrome. These obesity-related diseases include insulin resistance (type 2 diabetes), atherodyslipidemia (causing cardiovascular disease), hypertension (risk of stroke, heart attack and chronic renal failure) and fatty liver It is. The word “diabetes obesity” has been created due to the strong association between type 2 diabetes and obesity. Obesity is one of the most serious and prevalent health problems, affecting over 400 million people worldwide. Obesity rates in developing countries have tripled in the last 20 years, while Americans have the highest obesity rate in the world. 64% of US adults are overweight or obese, and about one third of US children and adolescents are overweight or obese. There is no shortage of “treatment” for obesity, including prescription and over-the-counter medications, weight loss programs, diets and exercise programs. Although some of these solutions are effective, they do not involve long-term improvements in weight or overall health, resulting in little to full expectations for both physicians and patients. Several prescription drugs act centrally to suppress appetite or block fat absorption (as an antinutritional agent). However, these therapies have limited efficacy and / or problems with various adverse events. Most patients using these treatments rebound and continue to gain weight.

  Obesity is not just the result of an unbalanced energy intake that exceeds consumption. In addition to the subject's genetic qualities, diet and exercise, the gut microbiota is the two dominant factors as the main environmental factor in the progression of obesity and metabolic syndrome (ie in genetic obese mice ob / ob mice) Changes in the gut microbiota (Ley et al., 2005) in the relative amount of various bacterial phylums (Bacteroides and Fermicutes) and increased ability to collect energy from the diet. Obesity traits are also contagious or infectious: sterile mice that have established the “obese microflora” produce a significantly greater increase in total fat than mice that have established the “slimming microbiota” (Turnbaugh et al. Meanwhile, sterile mice are resistant to high fat-induced diabetic obesity (Backhed et al., 2004; Backhed et al., 2007; Rabot et al., 2010). More importantly, lipopolysaccharide (LPS) produced by gram-negative bacteria in the gut microbiota plays an inductive role in diabetes obesity by “metabolic endotoxemia”: high fat diets contain LPS Not only the proportion of bacteria, but also LPS intestinal permeability is increased (Cani et al., 2007). Therefore, probiotics (Lee et al., 2006; Ma et al., 2008; Aronsson et al., 2010; Kadooka et al., 2010; Kang et al., 2010; Kondo et al., 2010; Chen et al Delzenne et al., 2011; Mozaffarian et al., 2011; Fak and Backhed, 2012; Ji et al., 2012; Teixeira et al., 2012) and prebiotics (Keenan et al., 2006; Known microbiota modulators / intestinal barrier enhancers that confer general health benefits to host animals, such as Zhou et al., 2008; Zhou et al., 2009), are identified in obesity and metabolic syndrome. It is promising as a new clinical tool in the therapeutic field. However, problems such as dietary changes that are undesirable for humans (ie, over 8% resistant starch prebiotics in the diet) can limit the use of these methods. However, while maintaining the longstanding concept of intestinal immune tolerance and weakening previous theories (Vijay-Kumar et al., 2010), anti-infectious congenitals controlled by pathogen pattern-sensitive toll-like receptors (TLRs) Immunity seems to be unable to prevent obesity and metabolic syndrome by overcoming tolerance and targeting the commensal microorganisms that cause obesity in the “obese microbiota” as foreign infectious microorganisms and inducing slimming microbiota It has recently been found that the basis for speculating the use of anti-infectious innate immune modulators as anti-diabetic obesity has been deprived (Ubeda et al., 2012).

  Currently, the only treatment that has proven effective in obesity is gastric bypass surgery. In normal digestion, food enters the small intestine through the stomach, where most of the nutrients and calories are absorbed; then it travels to the large intestine (colon), where the remaining waste is eventually excreted. In a typical Lewey stomach operation, a small sachet is made at the top of the stomach using surgical staples or plastic bands to make the stomach smaller; the small stomach is connected directly to the middle part of the small intestine (the jejunum) and the stomach Bypass the rest and the upper part of the small intestine (the duodenum). In addition to appetite suppression that can be maintained for up to 10 years and typical weight loss of 20-30 kg (Maggard et al., 2005), gastric bypass is type 2 within days after surgery, well before significant weight loss. Fully recovers from diabetes. However, gastric bypass surgery is due to high surgical costs, a markedly high mortality rate of about 1% due to complications, an unsuccessful rate of about 15%, irreversibility, gallstones, weight loss due to malabsorption and nutritional supplementation requirements. Limited to very serious persons. The basic anti-diabetic obesity mechanism of gastric bypass diverts undigested nutrients to the middle and lower gastrointestinal pathways, peptides YYY (PYY), glucagon-like peptide 1 (GLP-1) by nutrient-sensitive enteroendocrine cells , Oxyntomodulin (OXM), and cholecystokinin (CCK) are thought to stimulate the secretion of anti-diabetic anti-diabetic GI peptide hormones (Geraedts et al., 2009; Geraedts et al., 2010; Laferrere et al., 2010; Peterli et al., 2012). Attempts to use these anti-diabetic obesity hormones as injectable single therapies to treat obesity have been unsuccessful, probably due to the need to administer one or more hormones simultaneously (Field et al. , 2010). Interestingly, oral taste receptor cells show high functional similarity (in receptor expression and GI hormone production) to GI enteroendocrine cells (Wu et al., 2002; Dyer et al., 2005; Bogunovic et al. , 2007; Palazzo et al., 2007; Wang et al., 2009), and PYY delivered to the oral cavity of DIO mice probably interacts with specific Y2 receptors on afferent taste nerve fibers in the oral cavity. It was shown to induce a very good reduction in food intake and body weight through action (Acosta et al., 2011) (see also US 13 / 145,660). To date, there are no known drugs or methods that can stimulate the production of multiple GI hormones in oral taste receptor cells to confer an anti-diabetic obesity effect.

  Gastric bypass surgery and anti-nutritional methods (prone to adverse events) are mechanistically up-regulated in GI hormones such that anti-nutrients (such as zirlotapide) inhibit nutrient absorption and induce secretion of GI hormones. There is considerable overlap (Wren et al., 2007).

  Gastric bypass surgery and microbiota regulation / intestinal barrier-enhancing anti-diabetic obesity treatments (prebiotics and probiotics) also have considerable mechanistic overlap in the upregulation of GI hormones. Intestinal fermentation of non-digestible resistant starch, an anti-diabetic obesity prebiotic, releases short chain fatty acids resulting in sustained secretion of PYY and GLP-1 throughout the day (Keenan et al., 2006; Zhou et al ., 2008; Zhou et al., 2009). TLR2 agonist lipoprotein / lipopeptide (Sturm et al., 2005), TLR5 agonist flagellin (Schlee et al., 2007; Troge et al., 2012) and TLR9 agonist CpG DNA (Lammers et al., 2003; TLR agonists such as Menard et al., 2010; Zhong et al., 2012) are important contributors to the effective effects of probiotics. TLR agonists also have a non-immunomodulatory function that directly induces intestinal secretion of GI hormone (no activity has been reported to enhance anti-infectious innate immunity against pathogen load). Intestinal endocrine cells such as STC-1 cells express functional TLRs including TLR2 (Bogunovic et al., 2007). Activation of TLR4, TLR5 or TLR9 (by agonist LPS, flagellin, or CpG oligo DNA, respectively) induces secretion of GI hormone CCK from STC-1 enteroendocrine cells as well as in C57BL / 6 mice (Palazzo et al., 2007), suggesting a TLR-mediated anti-diabetic obesity gut endocrine mechanism for probiotics. In addition, saturated fatty acids are agonists of both TLR4 and TLR2 (Lee et al., 2001; Lee et al., 2004), so both these TLRs are most directly related to diabetes related nutrients in enteroendocrine cells. It plays the role of sensing nutrients. However, the possibility of this immune-independent TLR-GI hormonal pathway has not been investigated in the absence of new non-absorbable non-inflammatory TLR agonists, which is the traditional inflammatory TLR described above. This is because agonists must be ruled out as anti-diabetic obesity agents in order to prevent the possibility of adverse TLR-mediated systemic inflammatory responses (as mentioned above, LPS is actually a metabolic syndrome Can be induced). Accordingly, there is a need in the art for new therapeutic agents for obesity and metabolic syndrome.

  Brevibacillus texasporus (eg, ATCC PTA-5854) is a previously identified soil bacterium that expresses a non-ribosomal peptide synthase (NRPS, encoded by the operon with GenBank accession number AY953371) and has 13 amino acid residues A family / mixture of related cationic NRP variants ("BT peptide" or "BT lipopeptide" in light of their newly elucidated N-terminal structure, both terms interchangeable in this disclosure) Among them, BT1583 is the most abundant mutant (WO / 2005/074626). Cationic peptides (mixed or individual peptides separated from B. texasporus) show a broad spectrum of antibacterial activity in vitro (BT function No. 1). Due to the high 16S rDNA sequence identity (98.5%) between PTA-5854 and Brevibacillus laterosporus strains, Brevibacillus texasporus is classified as a subspecies of Brevibacillus laterospora, and Brevibacillus laterospora sp It is defined as a Brevibacillus laterosporus strain that produces a non-ribosomal peptide (or BT peptide) of origin. At least two types of B.I. This classification has been confirmed by genomic sequencing of Laterosporus strains (LMG15441 and GI-9). Both genomes (published under GenBank accession numbers AFRV00000000 and EMBL accession numbers CAGD01000001 to CAGD01000061, respectively) contain the complete BT NRPS operon with 99% DNA sequence identity with AY953371. Laterosporus strains are not known as producers of BT peptides. “Brevibacillus texasporus”, “Brevibacillus laterosporus subspecies texasporus” and “B. laterosporus” are thus synonymous.

The exact identity of the N-terminal residue of the BT peptide (including modifications thereof) is not known, and WO / 2005/074626 is a provisional N-terminal assignment of double methylated Bmt (4R) -4- [(E) -2-butenyl] -4-methyl-L-threonine, and Me ₂ -Bmt-Leu- _D Orn-Ile-Val-Val- _D Lys-Val- _D Leu-Lys, which is the overall structure of BT1583 _-D Tyr-Leu-Vol was shown. Here, Orn represents ornithine and Vol represents valinol. BT peptides have been discovered as antibiotics based on their antibacterial activity in vitro (WO / 2005/074626 and (Wu et al., 2005)), but orally delivered BT peptides (isolated from B. texasporus) As a mixture) lacks in vivo antimicrobial activity. For example, vancomycin-resistant enterococci (VRE) are highly sensitive to BT peptides in vitro, but BT peptides delivered orally at concentrations well above the minimum inhibitory concentration are unable to colonize VRE in the mouse GI pathway (Kogut et al. al., 2007). Importantly, orally delivered BT peptides are neither digested nor absorbed by the GI route, probably according to the presence of D-type amino acid residues and their relatively high molecular weight of about 1,600 daltons, respectively. Conceivable. However, orally delivered BT peptides (also as a mixture isolated from B. texasporus) can produce many effective systemic effects in animals. Orally delivered BT peptides are effective in preventing respiratory colibacillosis (air sac E. coli infection) and promoting growth and increasing feed efficiency in young chickens (Jiang et al., 2005). Perhaps more importantly, the in vivo anti-infective effect of the BT peptide appears to be independent of in vitro antibiotic activity, because orally delivered BT peptide is at a concentration below the in vitro minimum inhibitory concentration at E. coli and Salmonella. This is because it is effective in preventing infection by chickens (Jiang et al., 2005; Kogut et al., 2007; Kogut et al., 2009). It has also been discovered that circulating neutrophils and monocytes are primed (rather than activated) in BT ingested chickens, indicating innate immune regulation as a possible mechanism of action of these in vivo anti-infective effects . Given the fact that orally delivered BT peptide passes through the GI pathway without being digested or absorbed, it stimulates intestinal epithelial cells to secrete factors into the blood, which in turn primes leukocytes. It may increase anti-infectious immunity (BT function No. 2).

Bogolol is a family of five lipopeptide antibiotics (US 6,784,283) isolated from marine bacterium Brevibacillus laterosporus PNG276 found in a tubeworm in Papua New Guinea (US 6,784,283), which also includes a number of lipopeptide antibiotics tauramide. Produces other antibiotics (Gerard et al., 1999; Desjardine et al., 2007). Bogolol A, the most abundant variant, has a reported molecular weight of 1,584 and a reported structure (SEQ ID No: 1) Hmp- _E Dhb-Leu- _D Orn-Ile-Val-Val- _D It has Lys-Val- _D Leu-Lys- _D Tyr-Leu-Vol (Barsby et al., 2001; Barsby et al., 2006). Here, Hmp represents 2-hydroxy-3-methylpentanoic acid, and Dhb represents 2,3-dehydro-2-aminobutyric acid. As a pre-stage of the present invention, the present inventors have discovered that the BT peptide is a lipopeptide: BT1583 has the same structure as Bogolol A (SEQ ID NO: 1), and BT and the Bogolol family have four types Brevibacillus laterosporus PNG276 also shares a common NRP member. Texasporus strain (Examples 1 and 2).

  The present invention allows protease resistant BT peptides to induce physiological changes typically mediated by nutrient-sensing enteroendocrine / taste receptor cells and thus can be used as an oral anti-diabetic obesity pseudonutrient (BT function No. 3). This is based in part on the discovery of the present inventors. Orally delivered BT peptides have been shown to fully restore diet-induced hereditary obesity and insulin resistance in a pattern consistent with weight normalization rather than unidirectional weight loss.

  In one embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of one or more BT lipopeptides for treating or preventing obesity and diseases associated with obesity. By way of example, the BT lipopeptide of the invention has a sequence selected from SEQ ID NOs: 1-21. BT lipopeptide is a B.I. It can be made naturally (as a mixture or further purified individual peptides) or by chemical synthesis using a Texasporus strain. The BT lipopeptide of the present invention is provided as an isolated water-soluble lipopeptide, ie, a substantially purified form. “Substantially purified form” means that one or more peptides of the invention are at least about 1%, preferably at least about 10%, more preferably about 25%, even more preferably at least about 1% by weight of the composition. It constitutes about 50 wt%, even more preferably about 75 wt%, even more preferably about 95 wt%, and most preferably about 99 wt%. In one embodiment, the pharmaceutical composition of the present invention comprises B. An effective amount of BT peptide can be included as a mixture separated from Texasporus. The peptides of the present invention can be provided as salts, wherein these salts are acid or base addition salts depending on whether the portion of the peptide that binds to the salt (eg, amino acid side groups) is a basic or acidic moiety. Is included. Preferably, the salt is acceptable for pharmaceutical purposes. The invention also provides the peptides of the invention and their pharmaceutically acceptable salts in pharmaceutical compositions. Since the pharmaceutical composition of the present invention may contain a base, diluent or other material suitable for use in the pharmaceutical composition together with the peptide or the plurality of peptides, the BT peptide or the plurality of peptides of the present invention is substantially contained. May not necessarily be contained in a purified form.

  In one embodiment, the pharmaceutical composition of the invention for treating or preventing obesity and obesity-related diseases is in an oral dosage form, wherein an effective amount of water-soluble BT lipopeptide and peptide in the oral cavity of the subject. Ingredients that facilitate contact and / or exposure. In certain embodiments, the oral dosage form is an aqueous solution comprising one or more BT lipopeptides.

  In another embodiment, the invention relates to a method of inducing satiety in a subject, wherein a composition comprising one or more BT lipopeptides in a water-soluble dosage form is present in at least a portion of the subject's mouth, Including application in the previous period (before meals). The period may be 5 seconds or longer. In certain embodiments, the period is from 5 to 360 minutes before a meal. In a more specific embodiment, the period is 30 to 120 minutes before meals.

  Another embodiment is associated with a container containing a solid (eg, powder), liquid or semi-liquid composition comprising one or more BT lipopeptides in a water soluble form and a pharmaceutically acceptable base. In certain embodiments, the container includes a nozzle that discharges the composition into the mouth of the subject. The container may be under pressure and / or be equipped with a pump nozzle.

  Another embodiment relates to an article applicable to the mouth, to which one or more BT lipopeptides are added that are in a water soluble form. The article can be a chewing gum with one or more BT lipopeptides, a troche with one or more BT lipopeptides (eg, a soluble solid or semi-solid material intended to be held in the mouth for a period of time). Or a permeable pouch or sponge to which one or more BT lipopeptides are added. The article is designed for long-term delivery of one or more BT lipopeptides into the mouth and / or pharynx and includes conventional swallowing of pills, tablets or capsule compositions as is conventionally understood as swallowing orally. Contrary to In particular, the article is designed for delivery to the tongue.

  In certain embodiments, the one or more BT lipopeptides are delivered to the subject's mouth and / or pharynx according to a typical duration of at least 5, 10, 15 seconds or more. In another embodiment, delivery is between 0.1 and 120 minutes, including any specific increase by 0.1 minutes in such a range.

  In certain embodiments, the composition is prepared for spraying into the mouth. The pharmaceutical composition is placed in a container with a spray nozzle and can be released either by pumping or pressure release.

  In another embodiment, the pharmaceutical compositions are combined and provided in a chewing gum dosage form.

  In another aspect, the present invention provides a method for treating or preventing obesity and obesity-related diseases by oral administration of an effective amount of one or more BT lipopeptides. In one exemplary embodiment of the invention, an obese or overweight patient is selected and an amount of one or more BT lipopeptides is orally administered to the patient in an amount effective to reduce weight or weight gain. It is included. Another exemplary embodiment of the present invention is a method of treating or preventing type 2 diabetes, comprising an amount of one or more BT lipopeptides effective to control blood glucose levels and increase insulin sensitivity. Includes oral administration to patients. Another embodiment of the invention is a method of treating or preventing complex dyslipidemia, wherein the patient is provided with an amount of one or more BT lipopeptides effective to control blood LDL cholesterol and triglyceride levels. Oral administration is included. Another exemplary embodiment of the invention is a method of treating or preventing non-alcoholic fatty liver, one amount effective to treat or prevent fat accumulation in the liver and subsequent liver failure. This includes oral administration of the above BT lipopeptides to a patient. Yet another exemplary embodiment of the present invention is a method for treating or preventing hypertension, wherein an amount of one or more BT lipopeptides is orally administered to a patient in an amount effective to treat or prevent hypertension. Is included.

  In a further aspect, the present invention provides a method for reducing food intake, selecting obese or overweight patients and orally administering to the patient an amount of BT lipopeptide effective to reduce food intake. included.

  Preferably, the patient in the present invention is a human patient.

  The dose of BT lipopeptide as a non-absorbable pseudonutrient according to the invention is measured as mg of BT lipopeptide per kg dry weight of food consumption per day (mg per kg body weight per day). As contrary to the number). The present invention provides that an effective amount of BT lipopeptide pseudonutrient is in the range of 100-10,000 mg per kg dry weight of food consumed per day in single or divided doses.

  The present invention also provides a method for identifying a novel oral anti-diabetic obesity agent, identifying a non-inflammatory TLR agonist by in vitro screening using innate immune cells, and then orally delivering it to a diabetic obesity animal model Testing for indigestion, non-absorption and anti-diabetic obesity effects in (ie, DIO, db / db, or ob / ob mice). The present invention also provides a method for treating obesity and obesity-related diseases using such novel oral anti-diabetic obesity agents.

  Furthermore, the present invention relates to microorganisms (M. tuberculosis, Escherichia coli, Salmonella, Campylobacter, Staphylococcus aureus, Enterococcus faecium, Streptococcus pneumoniae, gonorrhea, Pseudomonas aeruginosa, Clostridium difficile, Acinetobacter baumannii, HIV, influenza, HBV, HCV, BT lipo as an oral adjuvant to enhance the immune response to vaccines, such as vaccines against herpesviruses, filoviruses, Epstein-Barr virus, HPV, Kaposi's sarcoma-associated herpesviruses, parasitic infections, cancers and combinations thereof A peptide is provided.

¹ H- ¹ corrected structure BT1583 showing the H NOE by a broken line Oral BT treatment with 40 ppm drinking water did not make a difference in weight gain, food or water intake in DIO mice. Oral glucose tolerance (oGTT) and glucose-induced insulin secretion in DIO mice were not altered by 30-day oral BT treatment with drinking water at 40 ppm. Oral BT treatment with drinking water at 400 ppm significantly reduced body weight, food and water intake in DIO mice. Oral glucose tolerance (oGTT) and glucose-induced insulin secretion in DIO mice were significantly improved by 30-day oral BT treatment with drinking water at 400 ppm. Body composition and hydration were significantly improved by 30-day oral BT treatment with drinking water at 400 ppm.

  In order to facilitate understanding of the invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as unspecified ("a", "an", etc.) and specific (such as "the") are not intended to imply a single element, but are specific examples that can be used in the description. Includes general categories. The terminology used herein is used to describe specific embodiments of the invention, but their use does not delimit the invention, except as set forth in the claims.

  “NRP variant” means a non-ribosomal peptide produced by a microorganism from the same NRPS. NRP variants may differ from each other in amino acid sequence at one or more residues depending on the extent of NRPS codon degeneracy in NRPS.

  By “non-inflammatory TLR agonist” is meant a TLR agonist that cannot elicit a response by itself in innate immune cells, but can increase the response of cells elicited by another stimulant. In other words, unlike traditional TLR agonists (such as TLR4 agonist LPS) that directly elicit inflammatory responses, the effects of non-inflammatory TLR agonists are only priming.

  “Pseudonutrients” can induce physiological changes typically mediated by nutrient-sensing enteroendocrine / taste receptor cells such as GI hormone secretion, anti-diabetic obesity effects, and enhanced nutrient absorption Means natural and synthetic compounds which are both sexual and non-absorbable. Pseudonutrients are distinguished from antinutrients (such as zirlotapide) that are natural or synthetic compounds that interfere with the absorption of nutrients that trigger the secretion of GI hormones (Wren et al., 2007).

  Effective to identify novel pseudonutrients capable of triggering secretion of anti-diabetic obesity GI hormone from enteroendocrine / taste receptor cells by pathways of immune-independent nutrient sensing other than TLR-mediated in enteroendocrine cells A two-step method is possible. The first step involves identifying non-inflammatory (priming but not activating) TLR agonists by screening compounds in vitro with innate immune cells such as isolated leukocytes. The second stage involves testing TLR agonists for indigestion, non-absorption and anti-diabetic obesity effects in DIO, db / db or ob / ob mice by oral delivery of TLR agonists. It is essential to exclude inflammatory TLR agonists such as LPS in order to avoid possible harmful systemic inflammation. The discovery of BT lipopeptide pseudonutrients provides a proof of concept for this screening method.

“Body Mass Index” (BMI) is a number based on an individual's weight and height and provides a way to assess the effect of weight on health. Kg value of individual and m value of elongation: BMI = kg / m ² . Healthy “normal weight” means that the BMI is 19-24.9. “Overweight” means BMI 25-29.9. “Obesity” means a BMI of 30 or more.

  “Administration” includes self-administration. “Oral delivery” or “oral administration” includes delivery or administration to the oral cavity and / or delivery or administration through the oral cavity.

  The BT peptides or BT lipopeptides used in the tests of Examples 3-7 do not use the reverse phase HPLC step to analyze BT NRP variants, and do not use WO / 2005/074626 and (Wu et al., 2005). Using the separation and detection methods published in It was a mixture of natural BT NRP variants isolated from Texaspos (including SEQ ID NO: 1-9). Those skilled in the art can easily prepare BT peptides or BT lipopeptides using the methods described in these references. The BT peptide or BT lipopeptide was characterized and the amino acid sequence is shown in Table 1 below. Table 1 shows the structures of nine BT NRP variants (beyond the detection level) and their typical relative amounts in such mixtures (non-BT-NRPS derived peptides were not detected by mass spectrometry) ).

Example 1
Mass spectrometry

  In WO / 2005/074626 and (Wu et al., 2005), the NRPS codon of the first module of the non-ribosomal peptide synthase of BT was predicted to be a threonine or dihydrothreonine (Dht) codon. The standard amino acid composition measurement of BT1583 did not provide evidence that either Thr or Dht was present as the first amino acid residue. Thus, the residue of Bmt could give a fragment whose double N-methylated form had a mass of 197 that roughly “matches” the 198 b-series m / z signals in tandem mass spectrometry (MS / MS). Was provisionally assigned only by.

  In order to better understand the N-terminal structure, the BT1583 peptide was further subjected to comprehensive MS / MS analysis, whereby 115.1 smaller b-series m / The z signal was shown as the mass of the first amino acid residue (Table 2). The only known amino acid residue with a mass of 83.0 Dalton is exactly dihydrothreonine (Dht), more commonly known in the literature as 2,3-dehydro-2-aminobutyric acid (Dhb) .

The identification of the first amino acid residue of BT1583 as Dhb raised the possibility that BT1583 and lipopeptide bogolol A have the same molecular structure. As a first test of this hypothesis, BT1583 was treated with HCl at 110 ° C. overnight to hydrolyze to free amino acids, and then subjected to MALDI-TDF mass spectrometry to determine whether BT1583 also contained Dhb and Hmp residues. In positive mode of detection, Tyr (M + H ⁺ /z=182.1), Lys (147.1), Orn (133.1), Leu ^/ Ile (132.1), Val (118.1) and Vol (11. 104.1) was clearly detected, but Dhb / Dht (102.1) or Thr (120.1) remained undetected. However, in the negative mode of detection, an M-H ⁺ / z signal of 131.1 was clearly detected, confirming the presence of the Hmp residue in BT1583.

  Eight additional BR NRP variants were separated by HPLC purification and were also subjected to the same comprehensive MS / MS analysis. All of these NRP mutants produced a b-series m / z signal of 115.1 in addition to 198.1 (Table 2), so they appear to contain the same N-terminal (Hmp-Dhb-) structure as BT1583. It was.

  Therefore, mass spectroscopic results revealed further structural similarity of the BT peptide with bogolol lipopeptide, and further studies were sought to determine the exact relationship between the two families of peptides.

Example 2
2D NMR

Two-dimensional NMR spectral analysis of the acetylated derivative of Bogolol A played an important role in the structural analysis of Bogolol A (US 6,784,283). In order to comprehensively elucidate the structure of natural BT1583, two-dimensional NMR spectral analysis of non-derivatized BT1583 was successfully performed. NMR data shows 13 amino acid residues (1 Dhb, 3 Leu, 1 Orn, 1 Ile, 3 Val, 2 Lys, 1 Tyr, 1 Vol) and 1 fatty acid in Hmp A 14-spin system corresponding to the residue was revealed. Complete resonance assignments were based on reported chemical shift values for amino acids, Hmp and Vol, and ¹ H- ¹ H total correlation spectroscopy (TOCSY), ¹ H- ¹ H correlation spectroscopy (COSY), ¹ H - ¹⁵ N performs a different species during one quantum coherence spectroscopy (HSQC) and ¹ H- ¹³ C cross species among multiple quantum coherence spectroscopy (HMQC) experiment (Table 3), was thereby be identified 14 spin system all . Nuclear overhauser enhanced spectroscopy (NOESY) map spectrum showed a cross peak due to dipole connectivity. NHi / NH (i + 1) and / or CαHi / NH (i + 1) cross peak, where i represents the amino acid position number, determined the amino acid sequence and the linkage of Hmp0 to Dhb1 at the N-terminus (FIG. 1).

In particular, in the olefin spectrum region, a quadruple signal (about ¹ Hδ = 5.58 ppm) having an intensity of 1: 3: 3: 1 was directly assigned to the Dhb1 Cβ proton. The (E) configuration assignment of Dhb1 was determined based on the NOE of the Dhb1 Cγ proton bound to the Leu2 NH proton.

The main features of Hmp0 were confirmed by TOCSY, COSY and HMQC experiments. A proton ( ¹ Hδ = 5.38 ppm) is identified as a hydroxyl proton based on its lack of binding to either carbon (in ¹ H- ¹³ C HMQC) or nitrogen (because δ is relatively low). It was. ^{In both 1} H- ¹ H COSY and ¹ H- ¹ H TOCSY, the hydroxy proton was found to interact only with the Hmp C2 proton ( ¹ Hδ = 3.59 ppm). The side group nature of Hmp at C3 was demonstrated by the exclusive COSY interaction of C6 methyl proton with C3 and also showed further COSY interaction with C2 and C4 protons. Finally, the NOE bond between the C2 and C6 protons was completely consistent with the L-Hmp residue.

Therefore, BT1583 and Brogor A have the same structure of Hmp- _E Dhb-Leu- _D Orn-Ile-Val-Val- _D Lys-Val- _D Leu-Lys- _D Tyr-Leu-Vol. In other words, B. BT1583 from Texasporus and its natural NRP variant are not N-terminally methylated as described and claimed in WO / 2005/074626. This conclusion is taken into account in the description of the natural BT NRP variant in Table 1 (for correlation with Bogolol, apparently excluding Bogolol E, an oxidized derivative of Bogolol D).

  Therefore, despite the large differences in natural habitat, Brevibacillus laterosporus PNG276 is also based on the production of its BT peptide family. It is a strain of Texasporus subspecies.

Example 3
BT and TLR2

  Orally administered BT lipopeptides prime (rather than directly activate) neutrophil and monocyte chicken blood innate immune cells (Kogut et al., 2007; Kogut et al., 2009) BT lipopeptides also prime isolated chicken neutrophils and monocytes in vitro (rather than directly activating) (Kogut et al., 2012). Since orally delivered BT lipopeptides are not absorbed into the blood and come into direct contact with these innate immune cells, priming leukocytes by BT in vitro never suggests that BT lipopeptides behave similarly in vivo. However, in vitro priming measurements are an effective tool to explore possible signaling pathways involved in BT-mediated innate immune regulation. Since TLR is a conserved receptor that plays an important role in innate immunity (Farnell et al., 2003) and TRL2 is the only TLR with lipopeptide agonists, the same in vitro BT priming measurement (Kogut et al. , 2012) to investigate whether TLR2 can function as a receptor for BT lipopeptides.

  Goat polyclonal antibody against human TLR2 was obtained from Santa Cruz Biotechnology Lab (Santa Cruz, CA). For TLR2 blocking, isolated chicken neutrophils and antibodies (2.0 μg / ml) were incubated at 39 ° C. for 30 minutes prior to treatment with BT and / or PMA.

  As already reported, treatment of neutrophils with BT lipopeptide (12 ppm) did not stimulate oxidative destruction in the absence of PMA (Table 4, Group 3 vs. Group 4), PMA-stimulated oxidative destruction was significantly increased about 3-fold (Group 4 vs. Group 2).

  Pretreatment of neutrophils with anti-human TLR2 goat antibody (2.0 μg / mL) reduced BT-primed oxidative destruction by 43% (Group 6 vs. Group 4), which indicates that these against chicken TLR2 The highest reported level of inhibition by human antibodies (Farnell et al., 2003). These results show that antibodies against human TLR2 at least partially inhibit BT neutrophil priming, TLR2 functions as a receptor for in vitro priming of purified BT lipopeptide, and BT lipopeptide It was demonstrated to be a non-inflammatory TLR2 agonist (priming only).

Example 4
BT lipopeptide as an oral adjuvant

  Because traditional inflammatory TLR2 agonists are used as adjuvants for various vaccines, the discovery of BT lipopeptides as non-inflammatory TLR2 agonists is a tradition for their in vivo immunomodulatory activity in mouse TB vaccine models Prompted the testing of effective adjuvant efficacy. C57BL / 6 mice are first treated with oral BT lipopeptide and then with one of two TB vaccines (a relatively weak ESAT-6 / MPL / DDA subunit vaccine and a strong BCG live vaccine) and then the vaccine Improved response to treatment was tested. The test group included:

  1. Oral BT lipopeptide treatment, administration for 3 days only at 40 ppm in drinking water.

  2. Oral BT lipopeptide treatment (40 ppm in drinking water) for 3 days followed by ESAT-6 / MPL / DDA injection at 1 × concentration on day 3.

  3. ESAT-6 / MPL / DDA injection only at 3.1 × concentration.

  4). Oral BT lipopeptide treatment (40 ppm in drinking water) for 3 days followed by MPL / DDA injection at 1 × concentration on day 3.

  5. Oral BT lipopeptide treatment (40 ppm in drinking water) for 3 days followed by BCG vaccine injection on day 3.

  6). BCG vaccine injection only.

  7). MPL / DDA injection only.

  8). Saline injection only.

  Mice were inoculated as described above and rested for 4 weeks before loading with a low dose aerosol (50-100 CFU) of the toxic Mycobacterium tuberculosis H37Rv. And the viable count was evaluated 30 days after the load. On the 30th day after infection, the number of viable bacteria was measured by homogenizing the organs of the mice and inoculating them on a 7H11 agar plate by serial dilution 10-fold.

  The results shown in Table 5 show that BT lipopeptide delivered orally at 40 ppm in drinking water for 3 days significantly increased the effect of ESAT-6 subunit vaccine (particularly in the target organ lung) to the level of live BCG vaccine Anti-infectious immunomodulatory activity in C57BL / 6 mice of oral BT lipopeptide at this dose of 40 ppm in drinking water (even when BT adjuvant was delivered separately from the vaccine) Was confirmed.

Example 5
BT-mediated immune regulation is not sufficient to confer anti-diabetic obesity activity

  The aforementioned findings that BT lipopeptides are TLR2 agonists are thought to reinforce the idea that BT peptides are responsible for obesity, because TLR2 signaling mediates diet-induced obesity (DIO) and metabolic syndrome in a mouse model (Ehses et al., 2010; Himes and Smith, 2010; Kuo et al., 2011). A DIO mouse study was performed to examine the effect of BT lipopeptides delivered orally at high immunomodulatory doses (at 40 ppm in drinking water as established by the traditional adjuvant study of Example 4) on obesity and metabolic syndrome It was.

  High fat diet-induced obese C57BL / 6 mice were used for evaluation of BT peptide effects. The mice were 18 weeks old and were fed on a high fat diet continuously for 12 weeks prior to treatment. The obesity level of the mice was moderate (weight approximately 40 g), and the effect of lipopeptide on body weight / obesity could be evaluated in both directions. In mice, Research Diets Inc. High fat diet D12492 (60% kcal% fat) obtained from Baseline measurements (weekly food and water intake, body weight, blood glucose, insulin, oral glucose tolerance test) were performed prior to treatment with native BT peptide. Randomly divided into 2 groups [control (non-peptide treated) and experimental (BT peptide treated) groups]. BT peptide was delivered at 40 ppm or 40 mg / L in drinking water. The effect of BT lipopeptide (on food and water intake, body weight, blood glucose level, insulin, oral glucose load) was evaluated.

  As shown in FIG. 2, oral BT treatment for 5 weeks with drinking water at 40 ppm did not produce a difference in weight gain, food intake, or water intake of DIO mice. Equivalent BT delivery concentration in food was kept constant at about 40 ppm (or mg per kg food) throughout the study.

  As shown in FIG. 3, 5-week oral BT treatment with drinking water at 40 ppm produced no difference in blood glucose control or insulin sensitivity in DIO mice.

  Orally delivered BT lipopeptides in an amount sufficient for effective in vivo modulation of anti-infectious innate immunity cannot alter diet-induced obesity or insulin resistance in either direction, supporting intestinal immune resistance (Lack of role of TLR-mediated anti-infective innate immunity in commensal-mediated control of diabetic obesity).

Example 6
Anti-diabetic obesity effect as a pseudo-nutrient of BT lipopeptide administered orally in DIO mice

  The discovery of BT lipopeptides as non-inflammatory TLR2 agonists is an unexpected and completely opposite expectation of their use as anti-diabetic obesity pseudonutrients by TLR-mediated nutrient sensing in enteroendocrine / taste receptor cells (“BT-enteric endocrine hypothesis). The threshold for inducing a GI hormone response is a minimum of 8% (or 80,000 ppm, 3 orders of magnitude higher than any BT concentration tested) resistant plasma in the diet. Because it is considered as high as required to increase GLP-1 and PYY (Belobrajdic et al., 2012), a new study of the potential anti-diabetic obesity effect in DIO mice has shown that BT concentrations ( The drinking water was increased by an order of magnitude from 40 ppm to 400 ppm.

  C57BLK / 6 DIO mice were 24 weeks old and were fed D12492 ad libitum for 18 weeks prior to treatment, and these mice were extremely obese and weighed approximately 50 g at the start of the study. Baseline measurements (weekly food and water intake, body weight, blood glucose, insulin, oral glucose tolerance test) were performed prior to treatment. A control group (10 animals) and a BT group (5 animals) were set. BT peptide was delivered at 400 ppm or 400 mg / L in drinking water for 30 days. The effect of BT lipopeptide (on food and water intake, body weight, blood glucose level, insulin, oral glucose load) was evaluated.

  As shown in FIG. 4, oral BT treatment with drinking water at 400 ppm resulted in significant weight loss in DIO mice. The constant weight loss began with BT administration and reached a maximum of about 15 g or 30% of the total body weight compared to the control around day 22. From day 22 to day 30, the average body weight of the BT group stabilized at about 35 g, which is the typical body weight (or normal body weight) of a 27-week-old healthy mouse fed a low-fat diet continuously. It remained. Therefore, BT completely restored DIO in these mice. It was also observed that the weight loss induced by BT stopped (and did not decrease too much) at that time when normal weight was reached, and the biological function of BT was rather than unidirectional weight loss. Suggested normalization of weight (optimization for ideal normal weight).

  The idea of weight normalization mediated by BT was also supported by changes in food intake. Initially oral BT treatment resulted in a stable drop in food intake, reaching a maximum of 40% on day 7. Thereafter (and for two weeks before full DIO recovery around day 22), food intake began to return and reached the amount of the control group by day 22. Appetite reduction and weight loss stopped at the same time as normal weight, so appetite suppression is the main driver of weight loss, and fine adjustment of appetite (in response to deviation from normal weight) It was clearly shown that it was the main mechanism.

  Water intake is reduced by about two-thirds throughout the study, so that equivalent BT food delivery concentrations are about 168, 218, 200, 186, 187 and 2 days, 7, 14, 22, and 30 days, respectively. It was 177 ppm (or mg per kg food). In other words, the actual dose in this study was about 5 times that of Example 5. Simultaneous suppression of food and water intake by orally delivered BT lipopeptide was the result predicted by the BT-enteric endocrine hypothesis. Because GLP-1 (Haak, 1999; Gutzwiller et al., 2004; Gutzwiller et al., 2006; McKay et al., 2011; Chan et al., 2013), PYY (Sloth et al., 2007) and CCK ( Koopmans et al., 1972; Guzek and Morawska, 1986; Verbalis et al., 1987; Bondy et al., 1989; Ebenezer, 1996; Menani and Johnson, 1998) can suppress water intake in addition to food intake. Because it is known. Interestingly, recalling “conditioned taste aversion” as an alternative explanation for the suppression of both water and food intake by BT would actually support the BT-enteric endocrine hypothesis. This is because conditioned taste aversion is mediated by GI hormones (Bojanowska, 2005; Chelikani et al., 2006; Schier et al., 2011) and oral taste cells, which mediate conditioned taste aversion.

  As shown in FIG. 5, oral BT treatment for 30 days at 400 ppm in drinking water caused a significant improvement in blood glucose control and an excellent enhancement of insulin sensitivity (> 4-fold).

  As shown in FIG. 6, oral BT treatment caused a significant improvement in body composition with about a 40% reduction in total fat mass and a minimal reduction in lean body mass.

  Importantly, oral BT treatment maintains body water content (both total and free water), and normal body hydration with reduced water intake results in increased internal water supply ("metabolism from lipolysis" Water ") and increased renal water retention (Guzek and Morawska, 1986; Verbalis et al., 1987), which was induced by the stimulation of the antidiuretic vasopressin by CCK. Oral delivery BT can be used to treat excessive thirst, fluid intake and urination associated with diabetes, as reduced fluid intake while maintaining body hydration is a pleasing advantage for diabetics. Adverse events due to reduced water intake that can occur in non-diabetic patients receiving oral BT can be easily avoided by the physician's instructions to add several cups of water daily.

  Furthermore, 30 days of oral BT treatment caused a visible reduction in visceral obesity and improvement in fatty liver in DIO mice. Importantly, no physiological or behavioral adverse events were observed throughout the study.

  Therefore, oral delivery of an effective amount of BT lipopeptide as a single agent (a “nutrient-sensing” amount) caused an excellent anti-diabetic obesity effect in DIO mice in a study aimed at mimicking nutrients.

Example 7
Anti-diabetic obesity effect as a pseudonutrient of BT lipopeptide delivered orally in db / db mice

  When BT lipopeptide was orally administered to db / db mice that are genetically obese and diabetic, the peptide produced a similar anti-diabetic obesity effect. Oral delivery of BT lipopeptide with drinking water at 400 ppm (average daily dose 15 mg / kg body weight) dramatically reduced body weight and improved glycemic control in db / db mice.

  However, once daily intragastric forced administration to db / db mice (15 mg of BT lipopeptide per kg body weight) does not produce such an effect, and BT lipopeptide is present in GI hormone-producing taste receptor cells. It was suggested that it is important to be in direct contact with or exposed to the oral cavity. Due to their cationic and amphiphilic properties, BT lipopeptides are known to be sticky to biomaterials and can be easily captured from the aqueous phase in vivo (US 61 / 447,703). When purified BT peptide is delivered orally as an aqueous solution, the free aqueous BT lipopeptide concentration can be lowered below the threshold to induce secretion of GI hormone from enteroendocrine cells due to the capture effect of the peptide by food or other gastrointestinal substances it is conceivable that.

  While this invention has been described in several embodiments, the present invention can be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations using the general principles of the present invention. Furthermore, this application is intended to cover deviations from this disclosure as it pertains to known or routine implementations in the art to which this invention pertains and which are within the scope of the appended claims.

All references, including publications, patents, and patent applications cited herein are individually and specifically indicated as if each reference was incorporated by reference, and all of which are described herein. As such, it is incorporated herein by reference within the same scope and includes the following references:


Acosta, A., Hurtado, MD, Gorbatyuk, O., La Sala, M., Duncan, D., Aslanidi, G., Campbell-Thompson, M., Zhang, L., Herzog, H., Voutetakis, A ., Baum, BJ, and Zolotukhin, S. (2011). Salivary PYY: a putative bypass to satiety.PLoS ONE 6, e26137.
Aronsson, L., Huang, Y., Parini, P., Korach-Andre, M., Hakansson, J., Gustafsson, JA, Pettersson, S., Arulampalam, V., and Rafter, J. (2010). Decreased fat storage by Lactobacillus paracasei is associated with increased levels of angiopoietin-like 4 protein (ANGPTL4) .PLoS ONE 5.
Backhed, F., Ding, H., Wang, T., Hooper, LV, Koh, GY, Nagy, A., Semenkovich, CF, and Gordon, JI (2004) .The gut microbiota as an environmental factor that regulates fat storage.Proc Natl Acad Sci USA 101, 15718-15723.
Backhed, F., Manchester, JK, Semenkovich, CF, and Gordon, JI (2007) .Mechanics underlying the resistance to diet-induced obesity in germ-free mice.Proc Natl Acad Sci USA 104, 979-984.
Barsby, T., Kelly, MT, Gagne, SM, and Andersen, RJ (2001) .Bogorol A produced in culture by a marine Bacillus sp. Reveals a novel template for protecting peptide antibiotics. Org Lett 3, 437-440.
Barsby, T., Warabi, K., Sorensen, D., Zimmerman, WT, Kelly, MT, and Andersen, RJ (2006) .The Bogorol family of antibiotics: template-based structure elucidation and a new approach to positioning enantiomeric pairs of amino acids. J Org Chem 71, 6031-6037.
Belobrajdic, DP, King, RA, Christophersen, CT, and Bird, AR (2012) .Dietary resistant starch dose-dependently reduces adiposity in obesity-prone and obesity-resistant male rats.Nutr Metab (Lond) 9, 93.
Bogunovic, M., Dave, SH, Tilstra, JS, Chang, DT, Harpaz, N., Xiong, H., Mayer, LF, and Plevy, SE (2007). Enteroendocrine cells express functional Toll-like receptors. Am J Physiol Gastrointest Liver Physiol 292, G1770-1783.
Bojanowska, E. (2005). Physiology and pathophysiology of glucagon-like peptide-1 (GLP-1): the role of GLP-1 in the pathogenesis of diabetes mellitus, obesity, and stress. Med Sci Monit 11, RA271-278 .
Bondy, CA, Jensen, RT, Brady, LS, and Gainer, H. (1989). Cholecystokinin evokes secretion of oxytocin and vasopressin from rat neural lobe independent of external calcium.Proc Natl Acad Sci USA 86, 5198-5201.
Cani, PD, Amar, J., Iglesias, MA, Poggi, M., Knauf, C., Bastelica, D., Neyrinck, AM, Fava, F., Tuohy, KM, Chabo, C., Waget, A. , Delmee, E., Cousin, B., Sulpice, T., Chamontin, B., Ferrieres, J., Tanti, JF, Gibson, GR, Casteilla, L., Delzenne, NM, Alessi, MC, and Burcelin, R. (2007) .Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 56, 1761-1772.
Chan, SW, Lin, G., Yew, DT, Yeung, CK, and Rudd, JA (2013). Separation of emetic and anorexic responses of exendin-4, a GLP-1 receptor agonist in Suncus murinus (house musk shrew) Neuropharmacology 70, 141-147.
Chelikani, PK, Haver, AC, and Reidelberger, RD (2006). Dose-dependent effects of peptide YY (3-36) on conditioned taste aversion in rats. Peptides 27, 3193-3201.
Chen, J., Wang, R., Li, XF, and Wang, RL (2011). Bifidobacterium adolescentis supplementation ameliorates visceral fat accumulation and insulin sensitivity in an experimental model of the metabolic syndrome. Br J Nutr 107, 1429-1434.
Delzenne, NM, Neyrinck, AM, Backhed, F., and Cani, PD (2011) .Targeting gut microbiota in obesity: effects of prebiotics and probiotics. Nat Rev Endocrinol 7, 639-646.
Desjardine, K., Pereira, A., Wright, H., Matainaho, T., Kelly, M., and Andersen, RJ (2007) .Tauramamide, a lipopeptide antibiotic produced in culture by Brevibacillus laterosporus isolated from a marine habitat: structure elucidation and synthesis. J Nat Prod 70, 1850-1853.
Dyer, J., Salmon, KS, Zibrik, L., and Shirazi-Beechey, SP (2005) .Expression of sweet taste receptors of the T1R family in the intestinal tract and enteroendocrine cells.Biochem Soc Trans 33, 302-305.
Ebenezer, IS (1996) .Systemic administration of cholecystokinin (CCK) inhibits operant water intake in rats: implications for the CCK-satiety hypothesis.Proc Biol Sci 263, 491-496.
Ehses, JA, Meier, DT, Wueest, S., Rytka, J., Boller, S., Wielinga, PY, Schraenen, A., Lemaire, K., Debray, S., Van Lommel, L., Pospisilik, JA, Tschopp, O., Schultze, SM, Malipiero, U., Esterbauer, H., Ellingsgaard, H., Rutti, S., Schuit, FC, Lutz, TA, Boni-Schnetzler, M., Konrad, D. , and Donath, MY (2010) .Toll-like receptor 2-deficient mice are protected from insulin resistance and beta cell dysfunction induced by a high-fat diet.Diabetologia 53, 1795-1806.
Fak, F., and Backhed, F. (2012) .Lactobacillus reuteri Prevents Diet-Induced Obesity, but not Atherosclerosis, in a Strain Dependent Fashion in Apoe-/-Mice.PLoS ONE 7, e46837.
Farnell, MB, Crippen, TL, He, H., Swaggerty, CL, and Kogut, MH (2003) .Oxidative burst mediated by toll like receptors (TLR) and CD14 on avian heterophils stimulated with bacterial toll agonists.Dev Comp Immunol 27 , 423-429.
Field, BC, Wren, AM, Peters, V., Baynes, KC, Martin, NM, Patterson, M., Alsaraf, S., Amber, V., Wynne, K., Ghatei, MA, and Bloom, SR ( 2010) .PYY3-36 and oxyntomodulin can be additive in their effect on food intake in overweight and obese humans.Diabetes 59, 1635-1639.
Geraedts, MC, Troost, FJ, Fischer, MA, Edens, L., and Saris, WH (2010) .Direct induction of CCK and GLP-1 release from murine endocrine cells by intact dietary proteins.Mol Nutr Food Res 55, 476 -484.
Geraedts, MC, Troost, FJ, and Saris, WH (2009) .Peptide-YY is released by the intestinal cell line STC-1.J Food Sci 74, H79-82.
Gerard, JM, Haden, P., Kelly, MT, and Andersen, RJ (1999) .Loloatins AD, cyclic decapeptide antibiotics produced in culture by a tropical marine bacterium.J Nat Prod 62, 80-85.
Gutzwiller, JP, Hruz, P., Huber, AR, Hamel, C., Zehnder, C., Drewe, J., Gutmann, H., Stanga, Z., Vogel, D., and Beglinger, C. (2006 ). Glucagon-like peptide-1 is involved in sodium and water homeostasis in humans.Digestion 73, 142-150.
Gutzwiller, JP, Tschopp, S., Bock, A., Zehnder, CE, Huber, AR, Kreyenbuehl, M., Gutmann, H., Drewe, J., Henzen, C., Goeke, B., and Beglinger, C. (2004). Glucagon-like peptide 1 induces natriuresis in healthy subjects and in insulin-resistant obese men. J Clin Endocrinol Metab 89, 3055-3061.
Guzek, JW, and Morawska, J. (1986) .Cholecystokinin affects water intake as well as neurohypophysial storage of vasopressin and oxytocin in the rat.Exp Clin Endocrinol 88, 200-206.
Haak, T. (1999) .New developments in the treatment of type 1 diabetes mellitus.Exp Clin Endocrinol Diabetes 107 Suppl 3, S108-113.
Himes, RW, and Smith, CW (2010). Tlr2 is critical for diet-induced metabolic syndrome in a murine model. FASEB J 24, 731-739.
Ji, YS, Kim, HN, Park, HJ, Lee, JE, Yeo, SY, Yang, JS, Park, SY, Yoon, HS, Cho, GS, Franz, CM, Bomba, A., Shin, HK, and Holzapfel, WH (2012) .Modulation of the murine microbiome with a concomitant anti-obesity effect by Lactobacillus rhamnosus GG and Lactobacillus sakei NR28. Benef Microbes 3, 13-22.
Jiang, YW, Sims, MD, and Conway, DP (2005) .The efficacy of TAMUS 2032 in preventing a natural outbreak of colibacillosis in broiler chickens in floor pens.Poult Sci 84, 1857-1859.
Kadooka, Y., Sato, M., Imaizumi, K., Ogawa, A., Ikuyama, K., Akai, Y., Okano, M., Kagoshima, M., and Tsuchida, T. (2010). of abdominal adiposity by probiotics (Lactobacillus gasseri SBT2055) in adults with obese tendencies in a randomized controlled trial. Eur J Clin Nutr 64, 636-643.
Kang, JH, Yun, SI, and Park, HO (2010) .Effects of Lactobacillus gasseri BNR17 on body weight and adipose tissue mass in diet-induced overweight rats.J Microbiol 48, 712-714.
Keenan, MJ, Zhou, J., McCutcheon, KL, Raggio, AM, Bateman, HG, Todd, E., Jones, CK, Tulley, RT, Melton, S., Martin, RJ, and Hegsted, M. (2006 Effects of resistant starch, a non-digestible fermentable fiber, on reducing body fat.Obesity (Silver Spring) 14, 1523-1534.
Kogut, MH, Genovese, KJ, He, H., Li, MA, and Jiang, YW (2007) .The effects of the BT / TAMUS 2032 optionally peptides on innate immunity and susceptibility of young chickens to extraintestinal Salmonella enterica serovar Enteritidis infection Int Immunopharmacol 7, 912-919.
Kogut, MH, Genovese, KJ, He, H., Swaggerty, CL, and Jiang, YW (2012) .BT BT peptides: small peptides that modulate innate immune responses of chicken heterophils and monocytes.Vet Immunol Immunopathol 145, 151-158 .
Kogut, MH, He, H., Genovese, KJ, and Jiang, YW (2009). Feeding the BT Cationic Peptides to Chickens at Hatch Reduces Cecal Colonization by Salmonella enterica Serovar Enteritidis and Primes Innate Immune Cell Functional Activity.Foodborne Pathog Dis.
Kondo, S., Xiao, JZ, Satoh, T., Odamaki, T., Takahashi, S., Sugahara, H., Yaeshima, T., Iwatsuki, K., Kamei, A., and Abe, K. ( Antiobesity effects of Bifidobacterium breve strain B-3 supplementation in a mouse model with high-fat diet-induced obesity. Biosci Biotechnol Biochem 74, 1656-1661.
Koopmans, HS, Deutsch, JA, and Branson, PJ (1972) .The effect of cholecystokinin pancreozymin on hunger and thirst in mice.Behav Biol 7, 441-444.
Kuo, LH, Tsai, PJ, Jiang, MJ, Chuang, YL, Yu, L., Lai, KT, and Tsai, YS (2011) .Toll-like receptor 2 deficiency improves insulin sensitivity and hepatic insulin signaling in the mouse. Diabetologia 54, 168-179.
Laferrere, B., Swerdlow, N., Bawa, B., Arias, S., Bose, M., Olivan, B., Teixeira, J., McGinty, J., and Rother, KI (2010). Rise of oxyntomodulin in response to oral glucose after gastric bypass surgery in patients with type 2 diabetes.J Clin Endocrinol Metab 95, 4072-4076.
Lammers, KM, Brigidi, P., Vitali, B., Gionchetti, P., Rizzello, F., Caramelli, E., Matteuzzi, D., and Campieri, M. (2003) .Immunomodulatory effects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cells. FEMS Immunol Med Microbiol 38, 165-172.
Lee, HY, Park, JH, Seok, SH, Baek, MW, Kim, DJ, Lee, KE, Paek, KS, and Lee, Y. (2006) .Human originated bacteria, Lactobacillus rhamnosus PL60, produce conjugated linoleic acid and show anti-obesity effects in diet-induced obese mice. Biochim Biophys Acta 1761, 736-744.
Lee, JY, Sohn, KH, Rhee, SH, and Hwang, D. (2001). Saturated fatty acids, but not unsaturated fatty acids, induce the expression of cyclooxygenase-2 mediated through Toll-like receptor 4.J Biol Chem 276 , 16683-16689.
Lee, JY, Zhao, L., Youn, HS, Weatherill, AR, Tapping, R., Feng, L., Lee, WH, Fitzgerald, KA, and Hwang, DH (2004) .Saturated fatty acid activates but polyunsaturated fatty acid inhibits Toll-like receptor 2 dimerized with Toll-like receptor 6 or 1.J Biol Chem 279, 16971-16979.
Ley, RE, Backhed, F., Turnbaugh, P., Lozupone, CA, Knight, RD, and Gordon, JI (2005). Obesity alters gut microbial ecology.Proc Natl Acad Sci USA 102, 11070-11075.
Ma, X., Hua, J., and Li, Z. (2008) .Probiotics improve high fat diet-induced hepatic steatosis and insulin resistance by increasing hepatic NKT cells.J Hepatol 49, 821-830.
Maggard, MA, Shugarman, LR, Suttorp, M., Maglione, M., Sugerman, HJ, Livingston, EH, Nguyen, NT, Li, Z., Mojica, WA, Hilton, L., Rhodes, S., Morton , SC, and Shekelle, PG (2005). Meta-analysis: surgical treatment of obesity. Ann Intern Med 142, 547-559.
McKay, NJ, Kanoski, SE, Hayes, MR, and Daniels, D. (2011). Glucagon-like peptide-1 receptor agonists suppress water intake independent of effects on food intake.Am J Physiol Regul Integr Comp Physiol 301, R1755- 1764.
Menani, JV, and Johnson, AK (1998) .Cholecystokinin actions in the parabrachial nucleus: effects on thirst and salt appetite.Am J Physiol 275, R1431-1437.
Menard, O., Gafa, V., Kapel, N., Rodriguez, B., Butel, MJ, and Waligora-Dupriet, AJ (2010). Characterization of immunostimulatory CpG-rich sequences from different Bifidobacterium species. Appl Environ Microbiol 76 , 2846-2855.
Mozaffarian, D., Hao, T., Rimm, EB, Willett, WC, and Hu, FB (2011) .Changes in diet and lifestyle and long-term weight gain in women and men.N Engl J Med 364, 2392- 2404.
Palazzo, M., Balsari, A., Rossini, A., Selleri, S., Calcaterra, C., Gariboldi, S., Zanobbio, L., Arnaboldi, F., Shirai, YF, Serrao, G., and Rumio, C. (2007) .Activation of enteroendocrine cells via TLRs induces hormone, chemokine, and defensin secretion.J Immunol 178, 4296-4303.
Peterli, R., Steinert, RE, Woelnerhanssen, B., Peters, T., Christoffel-Courtin, C., Gass, M., Kern, B., von Fluee, M., and Beglinger, C. (2012) Metabolic and hormonal changes after laparoscopic Roux-en-Y gastric bypass and sleeve gastrectomy: a randomized, prospective trial. Obes Surg 22, 740-748.
Rabot, S., Membrez, M., Bruneau, A., Gerard, P., Harach, T., Moser, M., Raymond, F., Mansourian, R., and Chou, CJ (2010). Germ- free C57BL / 6J mice are resistant to high-fat-diet-induced insulin resistance and have altered cholesterol metabolism. FASEB J 24, 4948-4959.
Schier, LA, Davidson, TL, and Powley, TL (2011) .Ongoing ingestive behavior is rapidly suppressed by a preabsorptive, intestinal "bitter taste" cue. Am J Physiol Regul Integr Comp Physiol 301, R1557-1568.
Schlee, M., Wehkamp, J., Altenhoefer, A., Oelschlaeger, TA, Stange, EF, and Fellermann, K. (2007). Induction of human beta-defensin 2 by the probiotic Escherichia coli Nissle 1917 is mediated through flagellin Infect Immun 75, 2399-2407.
Sloth, B., Davidsen, L., Holst, JJ, Flint, A., and Astrup, A. (2007) .Effect of subcutaneous injections of PYY1-36 and PYY3-36 on appetite, ad libitum energy intake, and plasma free fatty acid concentration in obese males. Am J Physiol Endocrinol Metab 293, E604-609.
Sturm, A., Rilling, K., Baumgart, DC, Gargas, K., Abou-Ghazale, T., Raupach, B., Eckert, J., Schumann, RR, Enders, C., Sonnenborn, U., Wiedenmann, B., and Dignass, AU (2005) .Escherichia coli Nissle 1917 distinctively modulates T-cell cycling and expansion via toll-like receptor 2 signaling. Infect Immun 73, 1452-1465.
Teixeira, TF, Collado, MC, Ferreira, CL, Bressan, J., and Peluzio Mdo, C. (2012). Potential mechanisms for the emerging link between obesity and increased intestinal permeability. Nutr Res 32, 637-647.
Troge, A., Scheppach, W., Schroeder, BO, Rund, SA, Heuner, K., Wehkamp, J., Stange, EF, and Oelschlaeger, TA (2012) .More than a marine propeller-the flagellum of the probiotic Escherichia coli strain Nissle 1917 is the major adhesin mediating binding to human mucus.Int J Med Microbiol 302, 304-314.
Turnbaugh, PJ, Ley, RE, Mahowald, MA, Magrini, V., Mardis, ER, and Gordon, JI (2006). An obesity-associated gut microbiome with increased capacity for energy harvest.Nature 444, 1027-1031.
Ubeda, C., Lipuma, L., Gobourne, A., Viale, A., Leiner, I., Equinda, M., Khanin, R., and Pamer, EG (2012). Familial transmission rather than defective innate immunity shapes the distinct intestinal microbiota of TLR-deficient mice.J Exp Med 209, 1445-1456.
Verbalis, JG, Richardson, DW, and Stricker, EM (1987) .Vasopressin release in response to nausea-producing agents and cholecystokinin in monkeys.Am J Physiol 252, R749-753.
Vijay-Kumar, M., Aitken, JD, Carvalho, FA, Cullender, TC, Mwangi, S., Srinivasan, S., Sitaraman, SV, Knight, R., Ley, RE, and Gewirtz, AT (2010). Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science 328, 228-231.
Wang, H., Zhou, M., Brand, J., and Huang, L. (2009). Inflammation and taste disorders: mechanisms in taste buds. Ann NY Acad Sci 1170, 596-603.
Wren, JA, King, VL, Campbell, SL, and Hickman, MA (2007) .Biologic activity of dirlotapide, a novel microsomal triglyceride transfer protein inhibitor, for weight loss in obese dogs.J Vet Pharmacol Ther 30 Suppl 1, 33- 42.
Wu, SV, Rozengurt, N., Yang, M., Young, SH, Sinnett-Smith, J., and Rozengurt, E. (2002). Expression of bitter taste receptors of the T2R family in the gastrointestinal tract and enteroendocrine STC -1 cells.Proc Natl Acad Sci USA 99, 2392-2397.
Wu, X., Ballard, J., and Jiang, YW (2005) .Structure and biosynthesis of the BT peptide antibiotic from Brevibacillus texasporus.Appl Environ Microbiol 71, 8519-8530.
Zhong, Y., Huang, J., Tang, W., Chen, B., and Cai, W. (2012). Effects of probiotics, probiotic DNA and the CpG oligodeoxynucleotides on ovalbumin-sensitized Brown-Norway rats via TLR9 / NF-kappaB pathway. FEMS Immunol Med Microbiol 66, 71-82.
Zhou, J., Martin, RJ, Tulley, RT, Raggio, AM, McCutcheon, KL, Shen, L., Danna, SC, Tripathy, S., Hegsted, M., and Keenan, MJ (2008). Dietary resistant starch upregulates total GLP-1 and PYY in a sustained day-long manner through fermentation in rodents.Am J Physiol Endocrinol Metab 295, E1160-1166.
Zhou, J., Martin, RJ, Tulley, RT, Raggio, AM, Shen, L., Lissy, E., McCutcheon, K., and Keenan, MJ (2009). Failure to ferment dietary resistant starch in specific mouse models of obesity results in no body fat loss.J Agric Food Chem 57, 8844-8851.

Claims (17)

A pharmaceutical composition which is a dosage form for delivery to the mouth and / or pharynx of a subject for treating or preventing obesity and / or type 2 diabetes, which induces normalization of body weight and / or blood glucose level An effective amount of the isolated and purified BT lipopeptide,
The separated and purified BT lipopeptide is water-soluble,
The isolated and purified BT lipopeptide comprises all three peptides having the sequence of SEQ ID NO: 1 to 3 .   The pharmaceutical composition according to claim 1, wherein the isolated and purified BT lipopeptide further comprises one or more peptides of a sequence selected from SEQ ID NOs: 4-9. The pharmaceutical composition according to claim 1, wherein the isolated and purified BT lipopeptide further comprises all six peptides of the sequence of SEQ ID NO: 4-9.   The pharmaceutical composition according to any one of claims 1 to 3, wherein the separated and purified BT lipopeptide is a mixture separated from Brevibacillus texasporus.   The pharmaceutical composition according to any one of claims 1 to 4, wherein the separated and purified BT lipopeptide is a synthetic product.   6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the amount ranges from 100 to 10,000 mg per kg dry weight of food consumption per day in single or divided doses.   The pharmaceutical composition according to claim 1, wherein the dosage forms are chewing gum, troches, and oral sprays. A method of treating or preventing obesity, type 2 diabetes and / or other diseases associated with obesity, which requires an effective amount of BT lipopeptides isolated and purified from Brevibacillus texaspors Delivering to the mouth and / or pharynx of a subject (except a human),
The separated and purified BT lipopeptide is water-soluble,
The isolated and purified BT lipopeptide comprises all three peptides having the sequence of SEQ ID NO: 1 to 3 . A method for reducing body weight or weight gain, which is isolated from Brevibacillus texasporus and purified BT lipopeptide in an effective amount in the mouth and / or pharynx of a subject (excluding humans) in need thereof Including delivering,
The separated and purified BT lipopeptide is water-soluble,
The isolated and purified BT lipopeptide comprises all three peptides having the sequence of SEQ ID NO: 1 to 3 . A method of reducing food intake, wherein BT lipopeptides isolated and purified from Brevibacillus texaspors are delivered in an effective amount to the mouth and / or pharynx of a subject (except humans) in need thereof Including
The separated and purified BT lipopeptide is water-soluble,
The isolated and purified BT lipopeptide comprises all three peptides having the sequence of SEQ ID NO: 1 to 3 . A method of treating or preventing type 2 diabetes, the BT lipopeptide isolated and purified from Brevibacillus texasporus, in an amount effective to control blood glucose and increase insulin sensitivity. Delivering to the mouth and / or pharynx of a subject in need (excluding humans),
The separated and purified BT lipopeptide is water-soluble,
The isolated and purified BT lipopeptide comprises all three peptides having the sequence of SEQ ID NO: 1 to 3 . A method for treating or preventing complex dyslipidemia, wherein BT lipopeptide isolated and purified from Brevibacillus texasporus is used in an amount effective to control blood LDL cholesterol and triglyceride levels. Delivering to the mouth and / or pharynx of a subject (excluding humans) in need of
The separated and purified BT lipopeptide is water-soluble,
The isolated and purified BT lipopeptide comprises all three peptides having the sequence of SEQ ID NO: 1 to 3 .   The method according to any one of claims 8 to 12, wherein the separated and purified BT lipopeptide further comprises one or more peptides of a sequence selected from SEQ ID NOs: 4-21.   The method according to any one of claims 8 to 12, wherein the separated and purified BT lipopeptide further comprises one or more peptides of a sequence selected from SEQ ID NOs: 4-9. The method according to any one of claims 8 to 12, wherein the separated and purified BT lipopeptide further comprises all six peptides of the sequence of SEQ ID NO: 4-9.   The method according to any one of claims 8 to 15, wherein the separated and purified BT lipopeptide is a mixture separated from Brevibacillus texasporus.   The method according to any one of claims 8 to 10, wherein the subject (excluding a human) is obese or overweight.

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